EP4452277B1 - Industrielles verfahren zur extraktion und reinigung von phospholipiden - Google Patents

Industrielles verfahren zur extraktion und reinigung von phospholipiden

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Publication number
EP4452277B1
EP4452277B1 EP22854172.8A EP22854172A EP4452277B1 EP 4452277 B1 EP4452277 B1 EP 4452277B1 EP 22854172 A EP22854172 A EP 22854172A EP 4452277 B1 EP4452277 B1 EP 4452277B1
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Prior art keywords
extract
subnatant
settling
stirring
purification
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French (fr)
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EP4452277A1 (de
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Mara Pittarello
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Fidia Farmaceutici SpA
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Fidia Farmaceutici SpA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/10—Phosphatides, e.g. lecithin
    • C07F9/103—Extraction or purification by physical or chemical treatment of natural phosphatides; Preparation of compositions containing phosphatides of unknown structure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present invention describes and claims a new industrial process for the extraction and purification of phospholipids from animal brain, particularly pure and with a high percentage content of phosphatidylserine (PS) and phosphatidylcholine (PC), especially enriched in PS, and the use of said phospholipids in the treatment of the pathology defined as "Long-Covid".
  • PS phosphatidylserine
  • PC phosphatidylcholine
  • Phospholipids represent a class of lipids that contain phosphate, they are amphipathic molecules as they have a hydrophilic polar head and a hydrophobic non-polar tail, and are the main constituents of animal and plant cell membranes.
  • sphingophospholipids and glycerophospholipids (or phosphoglycerides), the latter deriving from sn-glycerol-3-phosphate wherein the glycerol (CH 2 OH-CHOH-CH 2 OH) is esterified in position 3 with orthophosphoric acid (H 3 PO 4 ) and in position 2 with a fatty acid, whereas different classes of compounds can be bound in position 1.
  • fatty acids In membrane phospholipids, there are two types of fatty acids: saturated fatty acids in which all the carbon atoms are saturated, and unsaturated fatty acids in which one or more double bonds are present; the fatty acid in position 2 of the diacyl-phosphoglycerides of cell membranes is usually unsaturated.
  • orthophosphoric acid has a second esterification with an alcohol (an amino alcohol or an amino acid with an alcohol group or a sugar)
  • diacylphospholipids are therefore indicated with the prefix phosphatidyl - followed by the name of the esterified compound with the phosphate group (for example phosphatidyl serine wherein serine is the esterified compound with the phosphate group).
  • Phospholipids represent about 70-80% of the lipid weight of cell membranes, and the main ones are: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and sphingomyelin, which together form over 50% of all membrane lipids.
  • Phosphatidylinositol is a phospholipid present in smaller quantities which however plays a crucial role in the genesis of intracellular signals.
  • Sphingophospholipids contain a long-chain aminoalcohol instead of glycerol: in sphingosine (C18) the amino group is bound with an amide bond to the carboxyl group of a fatty acid (forming a compound called ceramide), whereas the hydroxyl group is bound with an ester bond to the orthophosphate, in turn esterified with an amino alcohol, usually choline, thus forming a compound called sphingomyelin or ceramide-1-phosphorylcholine; when the ceramide, on the other hand, is bound via the hydroxyl group to a monosaccharide, a cerebroside is obtained, if the ceramide is bound via the hydroxyl group to an oligosaccharide, a ganglioside is formed, both gligolipids being phosphorus-free.
  • Phosphatidylcholine PC is the major phospholipid of eukaryotic cells comprising 40 to 50% of membrane phospholipids: an important molecule for cell proliferation and division, it represents the main source of choline for cholinergic neurons which use it in the synthesis of the neurotransmitter acetylcholine; when the demand for choline exceeds the re-synthesis turnover of phosphatidylcholine, the composition of the neuronal membranes can undergo changes which are such as to negatively affect their integrity and therefore the vitality of the neurons involved.
  • Acetylcholine is the neurotransmitter that mediates important neuronal functions such as respiration, muscle contraction, heart rhythm, short-term memory, and most of the pre-ganglionic neurons of the Sympathetic and Parasympathetic system, as well as motor neurons, use this neurotransmitter.
  • Phosphatidylethanolamine represents from 20 to 50% of the membrane phospholipids of mammals, but it represents about 30-45% of brain phospholipids, whereas phosphatidylserine is, on the contrary, one of the phospholipids with the lowest concentration, present for only 2-10% of total phospholipids ( Vance JE.; Journal of Lipid Research; 2008; 49:1377-1387 ).
  • Phosphatidylethanolamine is found particularly in the white matter of cerebral nervous tissue, nerves and spinal cord, where it participates in membrane fusion during cell division cytokinesis; PE is also an important precursor/substrate in numerous and various biochemical and cellular physiological processes in mammals.
  • Phosphatidylserine PS is the main acid membrane phospholipid, capable of influencing the stability, fluidity and organization of cell membranes, it is mainly distributed in the internal part of plasma membranes where it interacts with cytoplasmic elements, proving to be capable of activating two important cellular enzymes such as sodium/potassium ATPase and protein kinase C.
  • Phosphatidylserine is perhaps the most widely-studied phospholipid due to its demonstrated capacity of both stimulating the release of dopamine from the dopaminergic terminals of the striatum, and activating the adenylate-cyclase enzyme of hypothalamic neurons, but above all for preventing the loss of dendritic spines of hippocampal pyramidal neurons linked to brain aging ( Advances in Behavioural Biology; Lecithin Ed. by Hanin I. and Ansell GB.; vol.33; 1987 ).
  • PS has been the subject of numerous clinical trials in the evaluation of the prevention and/or therapy of cognitive decline in elderly persons and/or Alzheimer patients and depressive disorders in general ( Cenacchi T. et al.; Aging Clin Exp Res; 1993; 5:123-133 ; Biggio G. et al. Minerva Psichiatrica; 2018; 59(1):1-10 ). It has also been demonstrated how phosphatidylserine can counteract the increase in the cortisol hormone in subjects subjected to stress, facilitating their functional recovery ( Monteleone P. et al.; Neuroendocrinology; 1990; 52(3):243-8 ).
  • Animal phospholipids are normally extracted from the membranes using solvents or mixtures of solvent, among which, in particular, chloroform, a recognized carcinogenic solvent, with subsequent purification by means of silica gel chromatography ( SU1,102,603 ; EP638083 ; US20120116104 ;); this solvent is also widely used in the purification of gangliosides from which the phospholipids are eliminated by chloroform-methanol partitioning ( EP0150712 ), or by saponification ( EP3095451 ).
  • solvents or mixtures of solvent among which, in particular, chloroform, a recognized carcinogenic solvent
  • CN 1 583 766 A discloses a process of extraction of PS from brain tissue.
  • WO 93/21190 A1 concerns the preparation of a phospholipid mixture comprising PS from bovine brain which is free from pathogenic agents.
  • the process comprises an extraction process and a purification method which is chosen from organic extraction or column chromatography.
  • US 3 436 413 also relates to the extraction of PL, in particular PS, from brain tissue.
  • the process comprises an extraction and washing steps and, in particular, it is examined the influence of the salts used on the distribution of the lipids in the different phases.
  • the review article of Biggio Giovanni et al.: "Overview of the pharmacological properties and therapeutic efficacy of phospholipid liposomes (Liposom Forte) in patients with depressive disorders” (MINERVA PSICHIATRICA, vol. 59, no. 1, 1 March 2018 (2018-03-01), pages 45-53 ) is a review article on the pharmaceutical properties and clinical trials of PL for treating depressive disorders.
  • EP 0 148 045 A1 relates to a phospholipid composition for treating CNS disorders, in particular disorders related to aging of brain.
  • the object of the present invention is to identify a process for the extraction and purification of animal phospholipids which overcomes the drawbacks of the state of the art, satisfies all the requirements listed above, and above all leads to the preparation of final PS-enriched phospholipids.
  • the present patent therefore relates to an innovative process for the extraction and purification of animal phospholipids as claimed in claim 1.
  • the Applicant has at its disposal a more effective phospholipid preparation in the treatment of all pathological conditions that require the administration of high concentrations of PS/PC, in particular PS, such as the prevention/treatment of problems of a neuroendocrine origin such as, for example, dementia and/or cognitive impairment in the elderly, and depressive disorders in general.
  • the present invention therefore further relates to phospholipids for use in the treatment of the pathology defined as "Long-Covid or Post-Covid", preferably phospholipids enriched in PS obtained according to the extraction and purification process object of the present invention for use in the treatment of the pathology defined as "Long-Covid or Post-Covid".
  • the disease Long-Covid affects a high percentage of patients who have recovered from the Sars-CoV-2 coronavirus and the pathologies caused by it, who unfortunately still show evident consequences of this infection; anosmia and ageusia, the persistent feeling of fatigue/asthenia (understood as the physical and/or mental decrease in the patient's performance), often headaches, sometimes serious psychiatric manifestations such as psychosis, are among the main neurological/neuroendocrine symptoms of Long-Covid, so-called "mental fog" is also frequently found, i.e., a state of mental confusion in which the patient who has recovered from the Sars-CoV-2 virus is unable to concentrate and have full control of his mental faculties, and therefore carry out the normal daily activities of his pre-Covid life ( Walitt B.
  • the use of phospholipids enriched in PS is therefore part of the treatment of the Long-Covid pathology for promoting/restoring the psycho-physical balance of the patient and reducing the symptoms of this pathology, above all improving mental fog, not only in adults but also in adolescents.
  • the Applicant claims an innovative industrial process for the extraction and purification of phospholipids from animal brain (preferably from the hypothalamic area and the cortical area), preferably swine, which does not contemplate the use of chloroform and a purification step by means of silica gel chromatography, which allows a particularly pure final phospholipid product to be obtained as it is not contaminated by unconventional animal viruses (above all from the viruses responsible for spongiform encephalopathy), free of solvent residues and protein components, with a high percentage content of PS and PC, but enriched above all in PS, wherein the final PS/PC ratio obtained at the end of the extraction and purification steps ranges from 0.8 to 1.6, thus proving to be radically modified with respect to the initial PS/PC ratio of the original animal tissue membranes from which these phospholipids are extracted and purified, as well as being different from the PS/PC ratio obtained using the extraction and purification methods known to the state of the art.
  • the phospholipid product obtained from the innovative process object of the invention consists of the phospholipids Phosphatidylethanolamine PE, Phosphatidic acid PA, Phosphatidylinositol PI, Phosphatidylserine PS, Phosphatidylcholine PC, Sphingomyelin SFM; the final product has only traces of two membrane glycosphingolipids such as Cerebroside (neutral glycolipid) and Sulfatide (glycolipid sulfate ester), also proving to be free of gangliosides and purified of any protein (and non-protein) contaminant and any solvent residue used.
  • Cerebroside neutral glycolipid
  • Sulfatide glycolipid sulfate ester
  • compositions comprising or consisting of PS-enriched phospholipids obtained with the above industrial extraction and purification process of phospholipids from animal brain, and their use in the prevention/treatment of problems of a neuroendocrine origin such as, for example, dementia and/or cognitive impairment of the elderly, and depressive disorders in general.
  • the PS-enriched phospholipids mentioned above can be associated with drugs and/or pharmacologically or biologically active agents such as, for example, steroids, non-steroidal antiinflammatory drugs, natural animal and/or vegetable extracts and/or vitamins, preferably vitamins of group B.
  • drugs and/or pharmacologically or biologically active agents such as, for example, steroids, non-steroidal antiinflammatory drugs, natural animal and/or vegetable extracts and/or vitamins, preferably vitamins of group B.
  • the invention further relates to phospholipids and the relative pharmaceutical compositions which comprise or consist of said phospholipids, for use in the treatment of the pathology defined as "Long-Covid".
  • PS-enriched phospholipids obtained with the industrial process for the extraction and purification of phospholipids from animal brain object of the invention (as defined above) for use in the treatment of the pathology defined as "Long-Covid", as the Applicant has surprisingly discovered, and subsequently demonstrated, that treatment with these phospholipids significantly improves, and also resolves, the symptoms of Long-Covid patients.
  • an antioxidant for example Oxynex ® LM
  • Oxynex ® LM can be conveniently added to the raw phospholipid extract.
  • the phospholipids were then analyzed for their identification by TLC chromatography stained with ninhydrin and/or copper sulfate as known to skilled persons in the field ( Bitman J. & Wood DL.; Journal of Liquid Chromatography; 1982; 5:1155-1162 ), the tests necessary for determining their purity from biocontaminants (all of the microbiological controls required for inj ectable products according to current Ph. Eur.) and pyrogens (Pyrogen Test, Ph. Eur. 2.6.8.) were then effected, from proteins ( Lowry et al.; J. Biol. Chem; 1951; 193(1):265-275 ) and from solvent residues (by gas chromatographic determination with the headspace technique vs reference solution, as known to skilled persons in the field).
  • the pharmaceutical composition in injection form is preferable.
  • step b the extraction step was then carried out, in particular in point b. the mixture of solvents consisting of acetone/methylene chloride/methanol was added to the ground product indicated above, in a ratio of ground product/mixture equal to 1:2.2 w/v, wherein the solvents were in a ratio of 1: 1 ,4:0.6 (v/v); the process is then continued with the subsequent steps described to arrive at the intermediate purification: step e.: the NaCl and KCl salts were added to the wet raw material of the previous step in quantities equal to 1.5 g/Kg of initial ground product; the process is then continued with the following steps f.-h.
  • step g wherein CaCl 2 dihydrate was added in a quantity equal to 1.6 g/L of extract; the process is then continued with step g. wherein 0.1L of acetone per L of subnatant produced was added, the process was then continued with step h., filtering the product by means of a filter press with a polypropylene filter obtaining the solid raw phospholipid.
  • the phospholipids were identified by TCL (Thin Layer Chromatography) stained with copper sulfate, it was therefore possible to compare the phospholipids present through their RF (Retardation Factors), i.e. based on the ratio between the distance covered by the phospholipid and the distance covered by the solvent, and they were found to be, in order (from the bottom): SFM, PC, PS, PI, PA and PE, with traces of cerebrosides and sulfatides; gangliosides absent.
  • RF Retardation Factors
  • the TCL was read with a photodensitometer at 450 nm:
  • Example 1 unequivocally demonstrates how the process for the extraction and purification of phospholipids from animal brain object of the present invention determines the production of substantially pure phospholipids enriched in PS.
  • Phospholipids prepared according to the process object of the invention (Example 1), formulated on an aqueous basis with mannitol and sodium phosphate as excipients, were administered to the patient intramuscularly at a dose of 28 mg/2 ml of injectable composition, for a daily treatment of 20 days.

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Claims (6)

  1. Verfahren zur Extraktion und Reinigung von Phospholipiden, die an Phosphatidylserin PS angereichert sind, wobei das endgültige PS/Phosphatidylcholin PC-Verhältnis am Ende der Extraktions- und Reinigungsschritte im Bereich von 0,8 bis 1,6 liegt, das die folgenden Schritte umfasst oder daraus besteht:
    Extraktion, die die folgenden Schritte umfasst:
    a. Zermahlen des tierischen Gehirns;
    b. Zugeben eines Gemisches von Lösungsmitteln, das Aceton und Methanol umfasst, zum gemahlenen Produkt;
    c. Rühren und Absetzen;
    d. Abtrennen des sedimentierten nassen Rohphospholipidextrakts;
    Zwischenreinigung, die die folgenden Schritte umfasst:
    e. Solubilisieren des aus Schritt d. erhaltenen nassen Rohphospholipidextrakts mit NaCl- und KCl-Salzen, Absetzen, Abtrennen des Subnatanten durch Filtration;
    f. Waschen des im vorherigen Schritt e. erhaltenen Subnatanten mit gereinigtem Wasser, Absetzen und Abtrennen des Subnatanten;
    g. Zugeben von Aceton und CaCl2-Dihydrat zu dem Subnatanten von Schritt f., Rühren und Absetzen;
    h. Filtrieren des Produkts von Schritt g. mittels einer Filterpresse, um einen ersten teilweise gereinigten festen Phospholipidextrakt zu erhalten;
    Endreinigung, die die folgenden Schritte umfasst:
    i. Solubilisieren des festen Phospholipidextrakts in einem organischen Lösungsmittel;
    j. Zugeben einer Lösung, umfassend EDTA, NaOH, KCl und NaCl mit einem pH-Wert von 8,5-9 zum Extrakt von Schritt i., Rühren und Absetzen;
    k. Abtrennen des Subnatanten von Schritt j., Zugeben von Wasser und Ethanol, Rühren und Absetzen;
    1. Abtrennen des Subnatanten von Schritt k. durch Filtration über einen Filter mit einem Filtrationsgrad von 3 µm oder alternativ 0,8 µm und anschließend 0,2 µm, Rühren und Absetzen;
    m. Waschen des Sediments aus Schritt 1. mit Aceton;
    n. Filtrieren des Produkts von Schritt m. unter Verwendung einer Filterpresse, um den gereinigten festen Phospholipidextrakt zu erhalten;
    o. Behandeln des endgültigen Extrakts von Schritt n. mit flüssigem Stickstoff, Granulieren und Vakuumtrocknen.
  2. Extraktions- und Reinigungsverfahren von PS-angereicherten Phospholipiden nach Anspruch 1, das die folgenden Schritte umfasst oder daraus besteht:
    Extraktion, die die folgenden Schritte umfasst:
    a. Zermahlen des tierischen Gehirns, vorzugsweise seines hypothalamischen Teils und/oder seiner Hirnrinde;
    b. Zugeben des Lösungsmittelgemisches, bestehend aus Aceton/Methylenchlorid oder einem anderen polaren organischen Lösungsmittel (POS)/Methanol zu dem gemahlenen Produkt von Schritt a., in einem Verhältnis (Kg/L) (gemahlenes Produkt) 1: (Gemisch) 1,5-3 G/V, vorzugsweise 1:2,2, G/V, wobei die Lösungsmittel des obigen Gemisches in einem Verhältnis von (Aceton): (Methylenchlorid oder einem anderen POS)1-2: (Methanol) 0,5-1 (V/V) stehen;
    c. Lassen mindestens 60 Minuten bei 30-35 °C unter Rühren; Absetzen des Extrakts für mindestens 180 Minuten bei 30-35 °C;
    d. Abtrennen des sedimentierten subnatanten Extrakts von Schritt c. durch Filtrieren über einen Filter mit einem Filtrationsgrad von 1 µm, vorzugsweise in Polypropylen, wobei der Überstand eliminiert wird;
    Zwischenreinigung, die die folgenden Schritte umfasst:
    e'. Abkühlen auf 0 °C und Solubilisieren des aus Schritt d. stammenden nassen Rohextrakts durch Zugeben von NaCl und KCl, vorzugsweise in einer Menge gleich 1-2 Gramm (pro Salzart)/kg des in Schritt a. erhaltenen gemahlenen Ausgangsprodukts und im Verhältnis 1:1 G/G, Rühren für mindestens 30 Minuten;
    e". Absetzen des Extrakts und Abtrennen des Subnatanten durch Filtrieren über einen Filter mit einem Filtrationsgrad von 1 µm, vorzugsweise in Polypropylen, wobei der Überstand eliminiert wird;
    f. Zugeben von gereinigtem Wasser zu dem Subnatanten von Schritt e" für mindestens 10 % seines Volumens, Lassen für mindestens 30 Minuten unter Rühren, Absetzen für mindestens 60 Minuten, Abtrennen des Subnatanten und Eliminieren des Überstandes;
    g'. Absenken der Temperatur auf 0 °C: Zugeben von Aceton zu dem Extrakt von Schritt f., vorzugsweise 1 L/L Extrakt, und CaCl2-Dihydrat, vorzugsweise 1-2 g/L Extrakt, Rühren und Absetzen für mindestens 30 Minuten, Entfernen des Überstands;
    g". Zugeben von Aceton zu dem Subnatanten von Schritt g'., vorzugsweise 0,1-0,2 L/L Subnatant, Rühren und Absetzen; dieser Waschschritt ist wiederholbar und wird bei 10 °C durchgeführt;
    h. Filtrieren des aus Schritt g". erhaltenen Produkts mittels einer Filterpresse, vorzugsweise mit einem Polypropylenfilter/-tuch (wobei der Überstand eliminiert wird), um einen ersten teilweise gereinigten festen Phospholipidextrakt zu erhalten;
    Endreinigung, die die folgenden Schritte umfasst:
    i. Solubilisieren des am Ende von Schritt h. erhaltenen festen Phospholipidextrakts in Methylenchlorid oder einem anderen POS, vorzugsweise in einem Verhältnis von 100-150 g Extrakt/L Lösungsmittel, Rühren für mindestens 60 Minuten lang bei 21-25 °C;
    j. Zugeben zu dem aus Schritt i. erhaltenen Extrakt, die Lösung, umfassend EDTA 110-130 Gramm/L, NaOH 7-9 Gramm/L, KCl 27-30 Gramm/L, NaCl 22-25 Gramm/L, hergestellt in gereinigtem Wasser und mit einem End-pH-Wert im Bereich von 8,5-9, in einer prozentualen Menge im Bereich von 21-24%, bezogen auf das Volumen des am Ende von Schritt i. erhaltenen Extrakts, und anschließend Ethanol in einer prozentualen Menge von 16-20%, wieder bezogen auf das Volumen des am Ende von Schritt i. erhaltenen Extrakts, Rühren für mindestens 60 Minuten und Absetzen für mindestens 12 Stunden;
    k. Abtrennen des Subnatanten und Eliminieren des Überstands; Zugeben von gereinigtem Wasser in Höhe von 10 % und Ethanol in Höhe von 1 % des Volumens des Subnatanten, Rühren für mindestens 60 Minuten und Absetzen für mindestens 12 Stunden;
    l. Abtrennen des Subnatanten vom Überstand (eliminiert) durch Filtration über einen Filter mit einem Filtrationsgrad von 3 µm, oder alternativ 0,8 µm, vorzugsweise in Polypropylen, dann 0,2 µm, vorzugsweise in Polytetrafluorethylen, Rühren und Absetzen, Abtrennen des Subnatanten und Eliminieren des Überstands;
    m. Zugeben von Aceton zu dem am Ende von Schritt 1 erhaltenen Sediment, vorzugsweise 0.5L/L des Subnatanten, Rühren für mindestens 30 Minuten bei einer Temperatur von 10 °C, Absetzen des Phospholipidniederschlags und Eliminieren des Überstands durch Absaugen; dieser Vorgang kann mehrmals wiederholt werden;
    n. Filtrieren des am Ende von Schritt m. erhaltenen Produkts unter Verwendung einer Filterpresse, vorzugsweise mit einem Polypropylenfilter/-tuch (wobei der Überstand eliminiert wird), um den gereinigten festen Phospholipidextrakt zu erhalten;
    o. Behandeln des aus Schritt n. erhaltenen endgültigen Extrakts mit flüssigem Stickstoff, Granulieren und Trocknen bei 40 °C mit einem Vakuum von :S 0,5 mbar.
  3. Extraktions- und Reinigungsverfahren von PS-angereicherten Phospholipiden nach einem der Ansprüche 1-2, das nicht die Verwendung von Chloroform und einen Reinigungsschritt durch Kieselgelchromatographie umfasst.
  4. Extraktions- und Reinigungsverfahren von PS-angereicherten Phospholipiden nach einem oder mehreren der Ansprüche 1-3, wobei das in Schritt a. verarbeitete Tiergehirn Schweinegehirn ist.
  5. PS-angereicherte Phospholipide, erhalten nach dem Extraktions- und Reinigungsverfahren nach einem oder mehreren der vorhergehenden Ansprüche 1-3, wobei das endgültige PS/PC-Verhältnis im Bereich von 0,8 bis 1,6 liegt.
  6. Pharmazeutische Zusammensetzungen, die PS-angereicherte Phospholipide nach Anspruch 5 umfassen oder daraus bestehen, zur Verwendung bei der Behandlung der als "Long-Covid" definierten Pathologie.
EP22854172.8A 2021-12-22 2022-12-19 Industrielles verfahren zur extraktion und reinigung von phospholipiden Active EP4452277B1 (de)

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US3436413A (en) * 1965-12-21 1969-04-01 Canada Packers Ltd Process for the isolation of phosphatidyl serine
IT1212900B (it) * 1983-11-17 1989-11-30 Valle Francesco Della Uso terapeutico della fosfatidilserina in malattie del sistema nervoso centrale senza effetti sulla coagulazione sanguigna
ES528692A0 (es) 1984-01-04 1985-07-01 Bioiberica Procedimiento para la obtencion de un complejo glicoesfingolipidico
IT1260149B (it) 1992-04-17 1996-03-28 Fidia Spa Metodo per la preparazione e purificazione di miscele di fosfolipidi prive di contaminanti da virus non convenzionali
CN1308334C (zh) * 2004-06-01 2007-04-04 山东师范大学 一种从动物脑提取磷脂酰丝氨酸的方法
US20090131368A1 (en) 2006-07-19 2009-05-21 Su Chen Mixtures of and methods of use for polyunsaturated fatty acid-containing phospholipids and alkyl ether phospholipids species
ES2750308T3 (es) 2015-05-22 2020-03-25 Bioiberica Proceso para preparar un extracto de cerebro de un animal

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IT202100032252A1 (it) 2023-06-22
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LT4452277T (lt) 2026-03-10
CN118475354A (zh) 2024-08-09

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